An Ex Vivo Explant Model for Studying Glial Interactions in the Mouse Retina

Summary

Here, we provide a detailed methodology for isolating the retina from the mouse eye for extended ex vivo experimentation. This protocol emphasizes making this technically demanding approach accessible for researchers who would like to take advantage of the research avenues afforded by keeping retinal glia in situ in live tissue.

Abstract

The role of glia in glaucoma is an increasingly prominent research topic, but much remains unknown about how populations of these support cells - namely astrocytes and microglia - influence retinal ganglion cell survival. While in vivo and in vitro models provide a degree of insight, both approaches have significant limitations, such as the impact of peripheral immune response in the former and changes to physiological function induced by cell isolation and culture in the case of the latter. To minimize these confounding factors, we have developed an ex vivo retinal explant system in which astrocytes, microglia, and other retinal cell types can be maintained in situ for periods of at least 3 days, enabling targeted investigation at a higher throughput than is typically feasible with in vivo models. Crucially, this approach is highly amenable to methodologies that would be challenging or unfeasible in a living animal yet remains compatible with common downstream assays of intact nervous tissue. Here, we present a protocol suitable for the isolation and culturing of intact retinal explants, along with representative results of immunofluorescence microscopy documenting the changes undergone by glia and retinal ganglion cells in the ex vivo retina.

Introduction

The light sensitive retina, an extension of the central nervous system (CNS) located at the back of the eye, is essential for sight but vulnerable to both acute injury and chronic disease. As with other CNS regions, neurons in the adult retina are not replaced when lost, and the retinal ganglion cells (RGCs) that aggregate and relay visual information to the brain are particularly vulnerable to dysfunction and death in glaucoma, resulting in irreversible vision loss^1,2. Glaucoma is a leading cause of blindness worldwide³, yet despite the devastating impact on affected individuals and th....

Protocol

All procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) at Schepens Eye Research Institute (Protocol # 2022N000030, approved 3/4/2025). Animals were handled in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.

NOTE: Figure 1 shows the key steps in the isolation of live retina from the enucleated mouse eye.

Results

At the macroscopic scale, explanted retinas remain essentially unchanged at both 1- and 3-day time points, although by the latter time they become relatively fragile, necessitating careful handling prior to fixation or other experimental endpoints. Retinas should be relatively flat, without folding (which can result in localized disruption to the diffusion of oxygen and nutrients), and should be suspended in the media between the air-liquid interface and the cell culture insert. By day 3, a faint streak may form on the b.......

Discussion

Organotypic culture³² approaches such as retinal explants harness the experimental flexibility and rapid turnaround time of cell culture while preserving much of the in situ context of in vivo studies, making them a potent avenue for investigating complex interactions between cell types. We present this protocol as an entry point for researchers who may have substantial experience with the eye and be comfortable manipulating a fixed retina but not a fresh one; therefore, we have .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#11 Scalpel blades	Bard-Parker	3,71,311
1 mL pipet ('Pipetman P1000')	Gilson	F144059M
100 mm Petri dish	Falcon	351029
16% Paraformaldehyde	Ted Pella Inc	18,505	Diluted to 4% for fixation
24 Well Plate	Falcon	353047	For fixation, blocking, and antibody incubation
35 mm Petri dish	Falcon	351008
6-well plate and insert kit	Greiner bio-one	657641
Alexa Fluor 488-Donkey Anti-Rabbit IgG (H+L)	Jackson ImmunoResearch Laboratories, Inc.	711-545-152	1:800 dilution
Alexa Fluor 594 Donkey anti-Rat IgG (H+L) Highly Cross-Adsorbed	Invitrogen	A-21209	1:800 dilution
Alexa Fluor 594-F(ab')2 Donkey Anti-Mouse IgG (H+L)	Jackson ImmunoResearch Laboratories, Inc.	715-586-150	1:800 dilution
Alexa Fluor 647-Donkey Anti-Guinea Pig IgG (H+L)	Jackson ImmunoResearch Laboratories, Inc.	706-605-148	1:800 dilution
Alexa Fluor 647-F(ab')2 Donkey Anti-Chicken IgY (IgG) (H+L)	Jackson ImmunoResearch Laboratories, Inc.	703-606-155	1:800 dilution
Angled forceps ('5/45')	FST / Dumont	11251-35
Anti-Brn3a antibody (mouse)	Chemicon	MAB1585	1:200 dilution
Anti-CD206 antibody (rat)	Biorad	MCA2235	1:200 dilution
Anti-GFAP antibody (chicken)	Abcam	ab4674	1:1000 dilution
Anti-Iba1 antibody (rabbit)	FUJIFILM Wako Pure Chemical Corporation	019-19741	1:500 dilution
Anti-Tmem119 antibody (guinea pig)	Synaptic Systems	400 004	1:500 dilution
B-27, 50x	Gibco (Thermofisher)	17504044
Blunt curved forceps ('Extra Fine Graefe)	FST	11152-10
Bovine Serum Albumin	Sigma-Aldrich	A9647	Use 1% w/v for blocking and antibody incubation
Filter kit (0.2 µm aPES membrane, 150 mL Bottle Top Filter)	fisherscientific	FB12566508
fine brush, size 3/0	princeton art & brush co.	06435-1030
GlutaMax	Gibco (Thermofisher)	35050061
Lab wipes (Kim wipes)	KIMTECH	34155
N-2, 100x	Gibco (Thermofisher)	17502048
Neurobasal-A Medium, minus phenol red	Gibco (Thermofisher)	12349015
Normal Donkey Serum	Jackson ImmunoResearch Laboratories, Inc.	017-000-121	Use at 10% for blocking and antibody incubation
Penicillin-Streptomycin	Gibco (Thermofisher)	15140122
Pipet Tips, 1000 µL	TipOne	1126-7810
Spring Scissors ('Cohan-Vannas')	FST	15000-11
Sterile water ('Dnase, Rnase free')	Invitrogen	10977-015
Sterile-filtered PBS	Gibco	10010-023
Straight forceps ('mini')	FST / Dumont	11200-14
Transfer Pipet, 5.8 mL	fisherscientific	13-711-9AMMD
Triton X-100	Thermo Scientific Chemicals	A16046.AP	Use at 0.5% for blocking and antibody incubation